FimX is a recently described protein in Pseudomonas aeruginosa (PA) shown to regulate type IV pill (TFP), an important virulence factor, in response to environmental signals. We propose to characterize the role of FimX, a GGDEF/EAL containing protein, in TFP assembly. We will confirm that FimX synthesizes cyclic di-GMP (c-diGMP), a regulatory molecule hypothesized to be important for a wide variety of cellular functions. The contributions of each FimX domain to regulation of c-diGMP will be elucidated. We have identified two components of ABC transporters that partner with FimX in a yeast two-hybrid screen. We will confirm these interactions with co-immunoprecipitation studies and identify the FimX domains required to bind these proteins. Furthermore, we will generate PA strains lacking these ABC transporters and confirm their role in the regulation of TFP. Additional studies will determine if these transporters are exporters or importers. These studies will determine whether c-diGMP is a second messenger regulating twitching motility in PA. We also will characterize the first ABC transporters identified in PA involved in controlling TFP expression.